Motor and sensory neurons of the periphery are among the largest cells, with volumes up to 5,000 times that typical for animal cells. Most of this volume is acquired just after stable synapse formation and concomitant with myelination, during which time the axons grow in diameter by more than an order of magnitude. One basic cell biological question is how this volume expansion, a key component for conduction velocity, is mediated. An essential feature of this is an "outside-in" signaling cascade from the myelinating cell that targets phosphorylation of the neurofilament subunit NF-M. Using gene replacement in mice to produce NFM that cannot be phosphorylated or that mimics phosphorylation, the effects of eliminating or chronically modifying this signaling target will be determined. This will include a focus on identification of putative interfilament crosslinkers that function to support survival and growth of motor neurons, including a search for components that may selectively bind to phosphorylated neurofilaments. In a second aim, a proportion of dominantly inherited ALS arises from mutation in superoxide dismutase (SOD1). Although we previously demonstrated that toxicity is non-cell autonomous, requiring damage generated in multiple cell types, we will now identify those cell types by selectively removing mutant SOD1 expression from individual cell types using Cre recombinase mediated gene deletion. As to the mechanism of intracellular damage, the role in toxicity from selective association of mutant SOD1 with spinal cord mitochondria will be determined. Mixed cultures of normal and mutant motor neurons and/or non-neuronal supporting cells will be used to identify how toxicity is developed non-cell autonomously. Laser capture microdissection and DMA arrays will be used to determine how motor neurons are damaged by mutant SOD1. Finally, following from our discovery of selective mutant association with spinal mitochondria, the mechanism for this and its role in toxicity will be determined with purified components.